Anti-inflammatory cranberry flavonol extract preparations

ABSTRACT

The present invention is directed to extracts of cranberries ( Vaccinium macrocarpon ) comprising either mixed flavonols that are substantially free of anthocyanins and proanthocyanidins or a purified cranberry flavonol compound, including myricetin-3-β-xylopyranoside, quercetin-3-β-glucoside, quercetin-3-α-arabinopyranoside, 3′-methoxyquercetin-3-α-xylopyranoside, quercetin-3-O-(6″-p-coumaroyl)-β-galactoside, and quercetin-3-O-(6″-benzoyl)-β-galactoside. The present invention also embodies the use of those extracts, as well as extracts comprising the cranberry flavonol compound quercetin-3-α-arabinofuranoside, for the treatment of inflammatory disorders. Pharmaceutical, food, dietary supplement, and cosmetic compositions utilizing the extracts or compounds of the present invention are also recited.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 USC §119(e) to U.S.Provisional Application Ser. No. 60/518,294, filed on Nov. 10, 2003,which is incorporated by reference in its entirety herein.

FIELD OF THE INVENTION

The present invention is directed to extracts of cranberries (Vacciniummacrocarpon) comprising either mixed flavonols that are substantiallyfree of anthocyanins and proanthocyanidins or a purified cranberryflavonol compound, including myricetin-3-β-xylopyranoside,quercetin-3-β-glucoside, quercetin-3-α-arabinopyranoside,3′-methoxyquercetin-3-α-xylopyranoside,quercetin-3-O-(6″-p-coumaroyl)-β-galactoside, andquercetin-3-O-(6″-benzoyl)-β-galactoside. The present invention alsoembodies the use of those extracts, as well as extracts comprising thecranberry flavonol compound quercetin-3-α-arabinofuranoside, for thetreatment of inflammatory disorders, particularly irritation of theurinary tract due to both bacterial and non-bacterial causes.

BACKGROUND OF THE INVENTION

Millions of women are diagnosed with urinary tract infections each year.Countless numbers of dogs and cats also suffer from chronic urinaryinfections and die from renal infections. E. coli is the most commonpathogen associated with these infections, causing over 80% of urinarytract infections. Over 30% of women suffer recurrent infections within a6 to 12-month period and are forced to resort to extended use ofantibiotics to treat these infections. Recurrent use of antibiotics canlead to pathogen resistance and result in deleterious side effects andtoxicity reactions. Consequently there exists a need for safealternative medications (e.g., non-antibiotics) that can be used toprevent or treat urinary tract infections in both animals and humans.Moreover, urinary tract inflammation is a painful and often debilitatingsymptom of bacterial infection as well as many diseases of both knownand unknown etiology. Thus, there is currently a need for new treatmentsto effectively mitigate the pain arising from inflammation of theurinary tract.

The two main forms of urinary tract infection are the renal infectionknown as pyelonephritis and the bladder infection referred to asbacterial cystitis. As the bladder is closer to the anus, the site ofentry for bacteria giving rise to urinary tract infections, cystitis isfar more common than pyelonephritis. Moreover, although cystitis is notas deadly as its renal counterpart, it is associated with pervasiveinflammation giving rise to severe bladder pain as well as frequent,urgent, and painful urination. Cystitis is also often a recurrentcondition, resulting in long term and often debilitating discomfort forthose afflicted.

Unfortunately, the causes of the battery of adverse conditions affectingthe urinary tract are only partially understood. Thus, inflammatoryconditions of the bladder of both known and unknown etiology arecommonly referred to as cystitis. As noted above, cystitis may bebacterial in nature, arising from infection of the bladder by E. coli.Cystitis may also embody urinary tract inflammation due to a number ofnon-bacterial sources, for example, allergic responses to food orinsufficient water intake, which allows the bladder and urethral tissuesto become dry, thus leading to deposition of crystallized uric acid onthe tissues and associated irritation. Interstitial cystitis is arecurrent condition of uncertain etiology. It is a source of frustrationfor both doctors and patients alike, as it has no apparent infectivecause and is frequently debilitating. Indeed, according to the firstepidemiological study of interstitial cystitis in the U.S. (Held, et al.1990), 50% of patients could not maintain full-time employment due tothe painful effects of the disease.

Treatments for cystitis include antimicrobials, anti-inflammatoryagents, buffering agents, muscle relaxants, mast cell stabilizers,painkillers including tricyclic antidepressants and transcutaneouselectrical nerve stimulator (TENS) units, catheterization andintravesicular instillation of heparin, the anti-inflammatory agentdimethyl sulfoxide, the detergent sodium oxychlorosene, the causticagent silver nitrate, or chromolyn sodium, and surgical techniques.Unfortunately, many of these treatments are themselves painful, and manyare also unsuitable for personal treatment in the home.

The fruit of the American cranberry (Vaccinium macrocarpon) has receivedconsiderable attention for its putative human health benefits. Most ofthe focus is on the flavonoid constituents due to their relatively highbiological activity in various assays. In vitro chemical assays haverated cranberries as having some of the highest antioxidant values ofover 21 fruits (Vinson et al. 2001; Sun et al. 2002), and the overallphenolic content appears to correlate with the level of antioxidantactivity. The ‘Folin-Ciocalteu’ colorimetric test has found cranberry tohave one of the highest phenolic contents of a number of fruit speciestested (Vinson et al. 2001; Sun et al. 2002). The phenolic classesidentified in cranberry include phenolic acids (Marwan et al. 1982;Heimhuber et al. 1990; Zheng et al. 2000; Zuo et al. 2002), anthocyanins(Hong et al. 1986; Hong et al. 1990), flavonols (Puski et al. 1967; Yanet al. 2002), and flavan-3-ols, which consist of both monomers and thepolymer classes procyanidins and proanthocyanidins (Foo et al. 1981; Fooet al. 2000a; Foo et al. 2000b; Cunningham et al. 2002). As describedmore fully below, we have previously identified cranberry A-typeproanthocyanidins as possessing anti-adherence activities againsturopathogenic type P E. coli (Foo et al. 2000a; Foo et al. 2000b; U.S.Pat. No. 6,608,102).

Cranberry juice has been shown to reduce bacteriuria associated withurinary tract infections in humans (Avorn et al. 1994). One mechanismimplicated in yielding this beneficial effect is the ability of certaincompounds present in cranberries to inhibit the adhesion of type I andtype P fimbriated E. coli to human epithelial cells (Sobota 1984;Schmidt et al. 1988; Zafriri et al. 1989). Type P fimbriated E. colihave been implicated as the main cause of pyelonephritis, while type Ifimbriated E. coli are the predominant causative agent of bacterialcystitis.

Zafriri et al. (1989) reported that fructose present in cranberry juiceinhibited the adherence of type 1 E. coli to uroepithelial cells, andthat cranberry juice also contained one or more non-dialyzablesubstances that inhibited binding of type P E. coli but failed to definethe chemical nature of those substances.

U.S. Pat. Nos. 5,474,774, 5,525,341, and 5,646,178 to Walker et al.disclose cranberry extracts having the ability to inhibit the adherenceof E. coli to uroepithelial cells. This activity was obtained byextracting whole cranberries with acidified alcohol followed byseparation of the activity from simple sugars by precipitation with ametal acetate or sulfate. Upon further manipulation, the reportedactivity consisted of a fraction enriched in polyphenol and flavonoidcompounds that contained as much as 10% anthocyanins. The specificity ofthis anti-adherence activity for type 1 or type P E. coli was, however,not determined.

WO 96/30033 and U.S. Pat. Nos. 5,646,178 and 5,650,432 to Walker et al.disclose a series of proanthocyanidin monomers, dimers, and polymers aswell as flavonoid derivatives thereof and related compounds purported tohave the ability to interfere with bacterial adherence to a surface. Thedimers and polymers of Walker were limited to compounds having B-typeinterflavanoid linkages. However, Walker failed to provide anyexperimental data correlating biological activity with a specificallyidentified compound. The extraction method involved alkalinizing a plantmaterial homogenate to a pH greater than 10 (a treatment which causesdegradation of proanthocyanidins) and precipitating the polyphenoliccompounds (together with other materials) by addition of alcohol. Thisprecipitate contained the proposed anti-adherence activity and wasfurther fractionated to yield the purified active compound. Using thisprocess with an aqueous solution of commercially available Ocean Spraycranberry powder, Walker reported obtaining a single active compound andpartially characterized the compound but failed to provide its chemicalstructure. The Walker assay methods could not distinguish betweenanti-adherence activities with respect to type 1 or type P E. coli, thusWalker was also unable to characterize the biological activity of thiscompound.

Commonly assigned U.S. Pat. No. 6,608,102 to Howell et al. disclosesplant proanthocyanidin extracts that are substantially free ofanthocyanins and flavonols and specifically inhibit the adherence oftype P E. coli to epithelial cells. The Howell '102 patent representsthe culmination of our work that conclusively demonstrated thatproanthocyanidins are the chemical agents in cranberries and otherplants that are responsible for this anti-adherence activity, and thatproanthocyanidins having at least one A-type interflavanoid linkage areparticularly potent agents for this activity. Later U.S. Pat. Nos.6,210,681 and 6,440,471 to Walker et al. disclose similarproanthocyanidin extracts.

Thus, we have previously determined that proanthocyanidins present incranberries exhibit anti-adherence activity with respect to the bindingof uroepithelial cells solely by type P fimbriated E. coli. However, asbladder infections are associated with type 1 fimbriated E. coli, theanti-adherence activity of these compounds is ineffective for thetreatment of bacterial cystitis. Although fructose in cranberry juicehas been found to possess an analogous anti-adherence activity withrespect to the binding of type 1 fimbriated E. coli to uroepithelialcells and is therefore theoretically effective at hindering bladderinfections, this discovery was made well over a decade ago, yet thereremains a substantial need for efficacious treatments for cystitis.

Cranberries and cranberry products have been used in the treatment ofurinary tract infections both alone and in conjunction with othertherapies. The basis for such treatments has traditionally implicatedthe antimicrobial properties associated with cranberries. Originally,this antimicrobial activity was thought to arise from acidification ofthe urine due to the intake of cranberry juice, resulting in anunfavorable environment for bacterial survival. Later, the focus shiftedto the bacterial anti-adherence activity of fructose andproanthocyanidins that has been elucidated in the aforementionedliterature. For example, Cystopurin, a Roche product for the treatmentof cystitis that is available in the UK, incorporates a cranberry juiceextract as an adjunctive therapy in a potassium citrate buffer designedto alleviate painful urination by neutralizing urinary acidity. However,as noted above, the only constituent of cranberries known to possessanti-adherence activity against the type 1 fimbriated E. coli commonlyinvolved in bacterial cystitis is fructose. Moreover, inflammation ofthe urinary tract may also arise from non-bacterial sources, in whichcase such anti-adherence activities are therapeutically ineffective.Finally, individuals having non-bacterial cystitis must exercise care inthe process of self-medication with cranberry products, as, depending onthe nature of the preparation, acids and other bladder irritants may bepresent that could result in aggravation of the inflammatory condition.

After bacterial adherence, internalization of type 1 E. coli by bladderepithelial cells represents another potential target for the treatmentof bacterial cystitis. Traditional thinking had considered uropathogenicE. coli to exist as extracellular pathogens within the urinary tract,despite the fact that transmission electron microscopy studies ofinfected rat and mouse bladders indicated that bladder epithelial cellscould internalize the pathogens in vivo (Fukushi et al. 1979; Mc Taggartet al. 1990). Although such internalization was initially regarded as ahost defense mechanism, Mulvey et al. (1998) suggested that it inuredbeneficially to the survival of the bacteria. Specifically, theydetermined that type 1 E. coli induced programmed cell death andexfoliation of bladder epithelial cells, however, pathogens could avoidexpulsion by such mechanisms by invading into deeper tissue. They alsospeculated that the frequency of recurrence of infection despiteantibiotic treatment could be linked to the persistence of bacteriawithin the cells of the bladder long after the death of extracellularpathogens by such treatments. This speculation was later confirmed bytheir discovery that a persistent reservoir of E. coli could beestablished by bacterial invasion of bladder epithelial cells, followedby intracellular replication and reemergence of the pathogens (Mulvey etal. 2001). Upon reemergence, the pathogens evade clearance viaexfoliation of the infected bladder cells by anchoring themselves andinvading into deeper, healthy cells that become exposed to the bladderlumen as a result of the exfoliation of cells from more superficiallayers. These internalized pathogens can persist in a latent state and,as the result of an as yet undetermined trigger, reemerge to causerecurrent infections.

Martinez et al. (2000) reported that the FimH element, an adhesinlocated on the tip of the type 1 pilus, mediates the bacterial invasionof human bladder epithelial cells. Specifically, they determined thatthe FimH element induces rearrangements of the host cell cytoskeletonresulting in a zippering effect by which the host cell engulfs thepathogen. Martinez also reported that these cytoskeletal rearrangementsrequired protein tyrosine phosphorylation and phosphoinositide 3-kinaseactivation, and that a quercetin derivative, LY294002 (Vlahos et al.1994), was a potent inhibitor of both of these activities andeffectively inhibited type 1 pilus-mediated bacterial invasion ofbladder epithelial cells in urinary tract infections. Tyrosine kinaseinhibitors, including both quercetin and myricetin, directly inhibitenzymes such as the hexose transporter GLUT1 via specific competitionfor the ATP binding site (Vera et al. 2001). Myricetin has been found tobe the most potent flavonol for inhibiting transport of methylglucoseand deoxyglucose, while iso-rhamnetin (3′-methoxyquercetin) was the mostpotent at inhibiting transport of dehydroascorbic acid (Hera et al.2001).

Quercetin has attracted much attention for its potential healthbenefits, and has been associated with numerous biological activities,including anti-inflammatory activities (Formica et al. 1995). Quercetinand related compounds inhibit a number of the processes associated withinflammation including lipopolysaccharide induced production of nitricoxide and tumor necrosis factor α (TNF-α) (Kawada et al. 1998; Wadsworthet al. 1999) and cytokine production (Xagorari et al. 2001). Theinvasion of bladder cells by type 1 E. coli has been shown to inducecytokine production by a lipopolysaccharide dependent mechanism(Schilling et al. 2001). Among fruit species, the cranberry contains oneof the highest concentrations of quercetin, ranging from 11 to 25 mg/100g of fresh fruit (Bilyk et al. 1986; Hakkinen et al. 1999). Quercetin ispredominantly found in a conjugated form with various sugars, and thesugar moiety may significantly influence its bioavailability andadsorption (Hollman et al. 1995; Hollman et al. 1999; Woffram et al.2002).

Quercetin has been used in the treatment of inflammatory conditionsassociated with the urinary tract. U.S. patent application Ser. Nos.757,358 and 848,187 to Katske, et al. disclose compositions and methodsfor the treatment of non-bacterial prostatitis and non-bacterialcystitis, respectively. These compositions comprise bioflavonoids havinga substantial percentage of quercetin that exhibit both anti-oxidativeand anti-inflammatory properties and a digestive enzyme such as bromelinor papain to increase the intestinal absorption of the bioflavonoidcomponent. The compositions are directed towards treating the painassociated with cystitis and prostatitis by an anti-inflammatorymechanism. However, substances such as papain are allergenic to manyindividuals, and these enzymes have caustic and corrosive effects on thedigestive mucous membranes.

Thus, there is presently a need for non-antibiotic treatments forurinary tract infections. There is also a need for a generalized therapyfor cystitis, whether bacterial or non-bacterial in origin, that isinnocuous and can be used for self-medication in the home. Accordingly,applicants have discovered that the cranberry flavonol compositions ofthe present invention advantageously possess superior anti-inflammatoryactivity. These compositions are amenable to use in the treatment ofinflammatory disorders, particularly those of the urinary tract arisingfrom both bacterial and non-bacterial sources. Moreover, thesecompositions can be formulated to contain one or more flavonols that caninhibit the invasion of uroepithelial cells by type 1 fimbriated E.coli. Advantageously, these compositions are also free from irritants ofthe bladder as well as the digestive mucous membranes. Thus, thecranberry flavonol compositions of the present invention embody natural,innocuous treatments that can be used beneficially for self-medicationby those afflicted with inflammatory conditions of the urinary tractgenerally, and cystitis particularly. The cranberry flavonolcompositions of the present invention are effective for and amenable tothe treatment of such inflammatory conditions regardless of whether suchconditions originate from bacterial or non-bacterial sources.

SUMMARY OF THE INVENTION

The present invention is directed to cranberry flavonol extracts thatare substantially free of anthocyanins and proanthocyanidins. Theseflavonol extracts have marked anti-inflammatory activity, and are thususeful in the treatment of inflammatory conditions generally, andparticularly in the treatment of urinary tract inflammation. Theinvention also provides a method of obtaining these flavonol extractsfrom cranberries (Vaccinium macrocarpon). The present invention is alsodirected to purified cranberry flavonol compounds, includingquercetin-3-O-(6″-benzoyl)-β-galactoside, which we have found to possessmarked anti-inflammatory activity, as well as methods to obtain thesecompounds from cranberries. Additionally, the present invention embodiesa processing method to increase the yield ofquercetin-3-O-(6″-benzoyl)-β-galactoside in cranberry powder. Finally,the invention relates to methods of preventing or treating inflammatoryconditions, particularly urinary tract inflammation from eitherbacterial or non-bacterial sources, in a mammal by administering acomposition comprising a flavonol extract of cranberries or a cranberryflavonol compound such as quercetin-3-O-(6″-benzoyl)-β-galactoside tothe mammal in an amount and for a time sufficient to prevent, reduce oreliminate inflammation and thereby lead to an amelioration or curing ofthe condition. Preferably the mammal undergoing treatment is a human,but the method is also applicable to animals, especially domesticatedanimals, such as cats and dogs, and livestock.

Pharmaceutical compositions are provided which comprise a flavonolcomposition, including pharmaceutically acceptable salts of any of theflavonol compounds, and a pharmaceutically acceptable carrier. Alsoprovided are pharmaceutical compositions comprising at least onecranberry flavonol compound, such asquercetin-3-O-(6″-benzoyl)-β-galactoside, including pharmaceuticallyacceptable salts thereof, and a pharmaceutically acceptable carrier. Insome instances, it may be preferable to provide the therapeutic dosagein the form of a food additive in a beverage such as a cranberryjuice-based beverage containing additional flavonols orquercetin-3-O-(6″-benzoyl)-β-galactoside. The invention also providesfood and dietary supplement compositions comprising a flavonolcomposition or at least one cranberry flavonol compound, such asquercetin-3-O-(6″-benzoyl)-β-galactoside, including pharmaceuticallyacceptable salts of any of these compounds, mixed with a consumablecarrier. Consumable carriers include, but are not limited to, livestockfeed, domestic animal feed and consumable food products, especiallycranberry containing food products, and are defined more fully in thedetailed description of the invention herein. Finally, the presentinvention also provides cosmetic compositions incorporating the activeagents disclosed herein. These pharmaceutical, food, dietary supplement,and cosmetic compositions are useful to prevent or treat inflammatoryconditions generally, and particularly urinary tract inflammation, andmore particularly cystitis.

BRIEF DESCRIPTION OF THE DRAWINGS

Table 1 provides chromatographic and mass spectral data for individualcomponents of Fraction 1 of the 60% methanol eluate from Sephadex LH-20chromatography of cranberry extract.

Table 2 provides NMR data for eight components of Fraction 1 of the 60%methanol eluate from Sephadex LH-20 chromatography of cranberry extract.

Table 3 provides data on the inhibitory effect of cranberry fractionsfrom Sephadex LH-20 chromatography on TPA-induced edema of mouse ears.

Table 4 provides data on the inhibitory effect ofquercetin-3-O-(6″-benzoyl)-β-galactoside (Cranberry peak 19 fromFraction 1 of the 60% methanol eluate from Sephadex LH-20chromatography) on TPA-induced edema of mouse ears.

FIG. 1 shows HPLC chromatograms of the flavonol glycosides extractedfrom fresh cranberries (60% methanol Fraction 1 after Sephadex LH-20column chromatography) and a cranberry solids powder (90MX) showing anincrease in the concentration of the compound corresponding to Peak 19(having retention time of approximately 45 minutes) upon processing toform the cranberry solids powder, under the following chromatographicconditions: Sorbax SB-C18 250 mm×4.6 mm i.d. column; eluate, 2% formicacid in methanol; flow rate, 1 ml min⁻¹; absorbance, 340 nm.

FIG. 2 is the HPLC chromatogram of Fraction 1 of the 60% methanol eluatefrom Sephadex LH-20 chromatography, with peaks numbered corresponding tothe compound numbers presented in Table 2, under the followingchromatographic conditions: Sorbax SB-C18 250 mm×4.6 mm i.d. column;eluate, 2% formic acid in methanol; flow rate, 1 ml min⁻¹; absorbance,340 nm.

FIG. 3 is the HPLC chromatogram of Fraction 2 of the 60% methanol eluatefrom Sephadex LH-20 chromatography under the following chromatographicconditions: Sorbax SB-C18 250 mm×4.6 mm i.d. column; eluate, 2% formicacid in methanol; flow rate, 1 ml min⁻¹; absorbance, 340 nm, wherein thepeak labeled M is myricetin and the peak labeled Q is quercetin.

FIG. 4 shows the chemical structures of eight flavonol glycosidesisolated from Fraction 1 of the 60% methanol eluate from Sephadex LH-20chromatography, specifically: myricetin-3-β-xylopyranoside (2),quercetin-3-β-galactoside (5), quercetin-3-β-glucoside (6),quercetin-3-β-arabinopyranoside (9), quercetin-3-α-arabinofuranoside(10), 3′-methoxyquercetin-3-α-xylopyranoside (15),quercetin-3-O-(6″-β-coumaroyl)-β-galactoside (16a), andquercetin-3-O-(6″-benzoyl)-β-galactoside (19).

FIG. 5 shows the proton NMR spectrum (bottom) of Cranberry peak 16a fromFraction 1 of the 60% methanol eluate from Sephadex LH-20 chromatographyand the trace extracted from the 6 Hz optimized IMPRESS-HMBC data set at166.7 ppm showing 2- and 3-bond coupling responses from the olefinprotons of the coumaroyl moiety and the galactose C-6″ protons to thesame ester carbonyl resonance.

DETAILED DESCRIPTION OF THE INVENTION

We have characterized the flavonol glycosides in spray-dried cranberrypowder and have isolated a cranberry extract containing mixed flavonolsthat is substantially free of both anthocyanins and proanthocyanidins.This extract exhibits a high degree of anti-inflammatory activity in theTPA-induced mouse ear edema assay and is rich in glycosylated quercetinderivatives. We have also isolated and characterized six flavonols notpreviously reported in cranberry, including the novel compoundquercetin-3-O-(6″-benzoyl)-β-galactoside, which we have found to be apotent anti-inflammatory agent in the TPA-induced mouse ear edema assay.Interestingly, we have also discovered that this compound is moreabundant in processed cranberry powder than in the fresh fruit itself(FIG. 1), and thus have determined means to generate the purifiedcompound in high yield.

The cranberry flavonol extracts and compounds of the present inventioncan be used in various food, dietary supplement, pharmaceutical, and,cosmetic formulations for the treatment and prevention of inflammatoryconditions generally. Moreover, as many of the extracts are both rich inglycosylated quercetin derivatives and free from bladder irritants, theycan be used advantageously for the treatment and prevention of cystitisarising from either bacterial or non-bacterial origins. An “inflammatorycondition” is defined herein as any condition or disease in whichinflammation is either symptomatic or plays a prominent role in thecondition or disease, including but not limited to: urinary tractinfection and/or inflammation, osteoarthritis, rheumatoid arthritis,cardiovascular diseases, dermatitis, cancer, diabetes, obesity, asthma,multiple sclerosis, and other diseases in which inflammation isinvolved.

Compositions comprising the extracts or compounds of the presentinvention can be formulated for administration as foods or dietarysupplements using one or more consumable carriers. A “consumablecarrier” is herein defined as any food, food ingredient, or foodadditive, or any excipient utilized for tabletting, encapsulation, orother formulation of an active agent for oral administration, whetherfor human or animal use. Specific additives are well known to those ofskill and are listed in places such as the U.S. Pharmacopeia. Fordietary supplements, the extract can be mixed according to methodsroutine in the art. Dietary supplements can be prepared in a variety offorms including, but not limited to, liquid, powder, or solid pillforms. In the present invention, the active agents can be administeredeither alone or in combination with other phytochemicals where combiningcompounds or extracts would lead to synergistic effects. Examples ofother phytochemicals which can be used in combination with the activeagents of the present invention include, but are not limited to,resveratrol and its hydroxylated and methoxylated analogs, rosemaryextract, green tea extracts, orange peel extracts, Mexican Bamboo, andHuzhang extracts. The active agents of the present invention can also beadded directly to foods and ingested as part of a normal meal. Variousmethods are known to those skilled in the art for addition orincorporation of such agents into foods.

Alternatively, compositions comprising these extracts can beadministered as conventional pharmaceuticals using one or morephysiologically acceptable carriers or excipients, referred to herein as“pharmaceutically acceptable carriers.” Pharmaceutical compositions canbe formulated for administration by any route including, but not limitedto, inhalation or insufflation (through mouth or nose), oral, buccal,parenteral, topical, vaginal, or rectal administration. The activeagents of the present invention can be applied topically to treat andprevent inflammatory conditions in either pharmaceutical or cosmeticapplications. Compositions for use in the present invention can also beadministered in the form of tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as bindingagents, fillers, lubricants, disintegrants, or wetting agents. Examplesof specific compounds for use in formulating tablets and capsules aredescribed in detail in the U.S. Pharmacopeia Tablets comprising theextract can also be coated by methods well known in the art. Liquidpreparations for oral administration can also be used. Liquidpreparations can be in the form of solutions, syrups or suspensions, ora dry product for reconstitution with water or another suitable vehiclebefore use. Such liquid preparations can be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents, emulsifying agents, non-aqueous vehicles, and preservatives.Again, specific additives are well known to those of skill and arelisted in places such as the U.S. Pharmacopeia. The compositions of thepresent invention can be formulated to provide controlled time releaseof the active agents. For buccal administration the extract can beformulated as a tablet or lozenge.

For administration by inhalation, compositions for use in the presentinvention can be delivered in the form of an aerosol spray in apressurized package or as a nebulizer, with use of suitable propellants.In the case of a pressurized aerosol, the dosage unit can be determinedby providing a valve to deliver a metered dose. For rectaladministration or vaginal administration, compositions for use in of thepresent invention can be formulated as suppositories, creams, gels, orretention enemas.

Parenterally administered compositions are formulated to allow forinjection, either as a bolus or as a continuous infusion. Formulationsfor injection can be prepared in unit dosage forms, such as ampules, orin multi-dose units, with added preservatives. The compositions forinjection can be in the form of suspensions, solutions, or emulsions, ineither oily or aqueous vehicles. They may also contain formulatoryagents such as suspending agents, stabilizing agents, and/or dispersingagents. The active ingredient may also be presented in powder form forreconstitution with a suitable vehicle before use. Specific examples offormulating agents for parenteral injection are found in the U.S.Pharmacopeia.

Freeze dried cranberry powder extracted with 80% aqueous acetonefollowed by ethyl acetate yielded a phenolic fraction with a high degreeof anti-inflammatory activity in the TPA-induced mouse ear edema assay.In order to optimize the resolution of compounds within the mainphenolic classes (phenolic acids, anthocyanins, flavonols, andproanthocyanidins), we assayed a number of analytical C18 reverse-phasecolumns, eluates and gradients to determine which of these combinationsprovided the best HPLC profile. As a result, we determined that uponSephadex LH-20 column chromatography of the ethyl acetate extract withsuccessive volumes of water, 60% aqueous methanol (v/v) (collected intwo fractions referred to herein as Fraction 1 and Fraction 2), 100%methanol, and 70% aqueous acetone (v/v) to separate the cranberrycomponents generally into the main phenolic classes, HPLC separation ofthese eluates on a Zorbax SB-C18 reversed phase column with a binarysolvent system (Solvent A, 2% aqueous formic acid; Solvent B, 20% formicacid in methanol) and gradient elution (Linear gradient of 5-25% B from0 to 5 min, linear gradient of 25-40% B from 5 to 25 min, isocraticelution with 40% B from 25 to 30 min, linear gradient of 40-95% B from30 to 45 min, and isocratic elution with 95% B from 45 to 50 min)provides optimal simultaneous resolution of cranberry phenolic acids,anthocyarins and flavonols.

After Sephadex LH-20 column chromatography of the ethyl acetate extract,we assayed the 60% methanol (Fraction 1 and Fraction 2 combined), 100%methanol, and 70% acetone eluates for anti-inflammatory activity via theTPA-induced mouse ear edema assay as described in Example 2 herein. Ofthese three, the 60% methanol eluate exhibited a significant doseresponse effect in reducing the weight of mouse ear edema by 34.3% and78.6% at 166 μg and 500 μg, respectively, as compared to the estimatedmean inflammation of the acetone and TPA control. HPLC analysis with PDAdetection indicated this fraction was composed primarily of phenolicswith maximum absorbance near 350-360 nm. We determined that Fraction 1of the 60% methanol eluate consisted of flavonol glycosides, while thelater-eluting Fraction 2 consisted of flavonol aglycones, predominantlymyricetin and quercetin. Further isolation and determination of thecomponents was undertaken as described herein. As expected, the 70%acetone eluate tested positive for proanthocyanidins with theHCl-vanillin assay.

Assay of Fraction 1 of the 60% methanol eluate using the chromatographicmethod according to the present invention afforded 22 peaks in contrastto the 9 reported previously in cranberry (Yan et al. 2002) (FIG. 2).Additionally, the method of the present invention is more effective forthe separation of individual flavonol glycosides within this class offlavonoids. The increased resolution provided by our method enabled usto identify additional flavonoids, six of which were structurallydetermined. Two of these newly identified compounds, 16a and 19, arevery rare acylated quercetin-galactosides.Quercetin-3-O-(6″-p-coumaroyl)-β-galactoside (16a) has only beenreported in Ledum palustre L. (Jin et al. 1999). We have isolatedquercetin-3-O-(6″-benzoyl)-β-galactoside (19) for the first time from anatural source. The increased resolution provided by our method enablesthe isolation of individual flavonol glycosides or aglycones present incranberries and thus also the preparation of bioactive compositionsincorporating one or more of these compounds.

Using the analytical methods of the present invention, we discoveredsome distinct differences between the flavonol glycoside profiles ofprocessed cranberry powder and fresh cranberries, the most pronounced ofwhich were the elevated contents of myricetin, quercetin, andquercetin-3-O-(6″-benzoyl)-β-galactoside after processing. Specifically,we evaluated the content of the latter compound in extracts of threecommercial cranberry cultivars (Stevens, Ben Lear, and Early Black) andfour samples of processed cranberry powder. We consequently determinedthat this component rose from an average of 1.27% (Range 0.16-2.28%) ofthe total flavonol fraction in fresh fruit to 3.44% (Range 2.50-4.02%)in processed powder extracts, as shown in FIG. 1. As described inExample 3 below, we found this compound to be a highly effectiveinhibitor of inflammation in the TPA-induced mouse ear edema assay.Thus, our invention also embodies a method for obtaining this compoundin high yield by isolating the compound from processed cranberrypreparations. Such processing entails the concentration of cranberryjuice by means known to those of skill in the art, including but notlimited to heating at atmospheric pressure or under vacuum, freezedrying, or a combination of these to obtain a cranberry preparationhaving a high solids content.

The invention is illustrated more fully by the following non-limitingExamples. Moreover, applicants' article “Characterization of Flavonolsin Cranberry (Vaccinium macrocarpon) Powder” J. Agric. Food Chem. 2004,52, 188-195 is incorporated by reference in its entirety herein.

EXAMPLE 1 Isolation and Characterization of Flavonol Glycosides andAglycones from Cranberry Powder

Reagents

All reagents were purchased from Fisher Scientific (PA) and Sigma (MO)and were of analytical or HPLC grade. Dimethylsulfoxide (DMSO)-d₆(99.96% D) was obtained from Cambridge Isotope Laboratories (MA).Standards of quercetin-3-galactoside, quercetin-3-glucoside,quercetin-3-rhamnoside, quercetin, myricetin and kaempferol werepurchased from INDOFINE Chemical Company, Inc. (NJ). Sephadex™ LH-20 forcolumn chromatography was obtained from Amersham Pharmacia Biotech AB(Sweden).

Extraction and Fractionation of Flavonoids

Flavonoids from 1.0 kg of freeze dried cranberry powder 90-MX suppliedby Ocean Spray Cranberries, Inc., (MA) were extracted twice with 80%acetone/water (v/v) (1:10), filtered and partially evaporated underreduced pressure at 35° C. to remove the acetone. The resultant aqueousphase was defatted by extraction with hexane (1:1) and the aqueous layerwas further extracted with three portions of ethyl acetate (1:1). Afterconcentration in vacuo, the pooled ethyl acetate fraction (24.8 g) wasfurther fractionated using column chromatography. A portion of ethylacetate extract (8.0 g) was dissolved in 60% methanol and loaded ontothe 100×50 mm column packed with hydrated Sephadex LH-20. Subsequentelution with water, 60% methanol/water (v/v), 100% methanol and 70%acetone/water (v/v) was applied to fractionate phenolic classes andremove all phenolic constituents from the column. Fractions eluted weremonitored by analytical HPLC and tested for anti-inflammatory activityas described below. The 60% methanol fraction contained mostlyconstituents with maximum absorbance at 340 nm and was eluted from thecolumn in two sequential 1200 ml volumes, yielding 660 mg (Fraction 1)and 600 mg (Fraction 2) of phenolics. Fraction 1 was used for thefurther isolation of individual constituents by preparative HPLC asdescribed below. Final purification of the compound 16a was achieved ona Sephadex LH-20 column (20×40 mm) using methanol as the elutingsolvent.

HPLC Apparatus and Chromatographic Conditions

Analytical HPLC

HPLC analysis was performed on a Waters Millenium HPLC system composedof a Water In-line Degasser, a Waters 600E Multisolvent Delivery System,a Waters 717 plus Autosampler, and a Waters 996 photodiode arraydetector. A Zorbax SB-C18 250 mm×4.6 mm i.d. (5 μm) reversed phasecolumn protected with a Waters Guard-Pak Precolumn Module was used foranalysis. Separations were carried out in a binary solvent system:solvent A, 2% formic acid; solvent B, 2% formic acid in methanol. Aprogram of a linear gradient 5-25% B from 0 to 5 min, 25-40% B from 5 to25 min, an isocratic elution with 40% B from 25 to 30 min, a lineargradient 40-95% B from 30 to 45 min and an isocratic elution with 95% Bfrom 45 to 50 min at flow rate of mL/min was used. PDA detection wasused to monitor the eluate from 210 to 700 nm.

Preparative HPLC

A Zorbax SB-C18 250 mm×21.2 mm i.d. (5 μm) column was used with thebinary solvent system and gradient elution as described in the precedingsection and a flow rate of 15 mL/min. The column effluents weremonitored from 210 to 400 mm. Fractions were collected using a WatersFraction Collector II. A gradient elution program afforded thecollection of eight individual compounds that were purified byre-chromatography under the same conditions.

Identification of Cranberry Flavonol Glycosides and Aglycones

Individual constituents of the flavonol extract according to the presentinvention were identified by comparison of chromatographic retentiontime and UV spectral characteristics with standards, as well as by MSand NMR techniques.

Comparison with Standards

Standard curves for identification of flavonols were prepared usingauthentic standards dissolved in methanol at a concentration of 1 mg/mland stored at −20° C. as stock solutions. Identification ofquercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-rhamnoside,myricetin and quercetin was performed by matching their retention timeand spectral characteristics measured at 340 nm against those ofstandards.

Mass Spectrometry

Atmospheric pressure chemical ionization (APCI) mass spectrometry in thenegative-ion detection mode was obtained on a VG Platform massspectrometer (Micromass, Manchester, U.K.). A Zorbax SB-C18 250×4.6 mmreversed-phase column and the methanol/formic acid/water mobile phasedescribed above were used with a flow rate of 1 ml/min. Typical tuningparameters were as follows: corona, 3 kV; high voltage lens, 0.0 kV;cone 15 V; source temperature, 150° C.; and APCI probe temperature, 450°C. Spectra were scanned over a mass range of m/z 150-1100 at 1.0 s percycle.

Positive-ion electrospray ionization (ESI) mass spectrometry wasacquired on a ThermoFinnigan TSQ-Quantum mass spectrometer usingstandard operating parameters. A Zorbax Eclipse XDB C18 150×4.6 mmreversed-phase column and a binary solvent gradient of A(water/acetonitrile/trifluoroacetic acid (TFA) 95:5:0.025) and B(water/acetonitrile/TFA 5:95:0.025) were used. Spectra were scanned overa mass range of m/z 190-800 at 1.0 s per cycle.

NMR Spectroscopy

The NMR data were obtained on either a Varian INOVA three-channel NMRspectrometer operating at a ¹H observation frequency of 599.730 MHz andequipped with a 3 mm Nalorac Z•SPEC MIDTG gradient inverse tripleresonance NMR probe or a Varian INOVA three-channel NMR spectrometeroperating at a ¹H observation frequency of 499.792 MHz and equipped witha 5 mm Varian Chili-probe® gradient inverse triple resonance NMR probeoperating at a coil temperature of 25° K. The sample temperature wasregulated at 20° C. for all samples except peak 9, for which data wereacquired at 32° C. Samples were dissolved in −150 μL of DMSO-d₆ andtransferred to a Wilmad 3 mm NMR tube for analysis. Peak 16a wasdissolved in ˜150 μL of 90:10 DMSO-d₆:benzene-d₆. Chemical shifts werereferenced relative to the residual solvent resonances at 2.49 and 39.5ppm for ¹H and ¹³C, respectively. All ¹H NMR data were acquired with aspectral width of 16 ppm. Correlated Spectroscopy (COSY) data wereacquired as 256 increments with 8 transients per increment; squaredsinebell apodization was used in both dimensions. Heteronuclear SingleQuantum Coherence (HSQC) and Heteronuclear Single Quantum CoherenceTotal Correlation Spectroscopy (HSQCTOCSY) data were acquired as 96increments with 24 and 96 transients per increment, respectively. Bothdata sets had an F1 spectral window of 146 ppm and were apodized with agaussian weighing function in both dimensions; the HSQCTOCSY mixing timewas set to 18 ms. Heteronuclear Multiple Bond Correlation (HMBC) datawere acquired as 96 increments with 320 transients per increment. The F1spectral width was 241 ppm. Squared sinebell apodization was used inboth dimensions.

Results

Chromatography

FIG. 2 shows the chromatogram of Fraction 1 of the 60% methanol eluatefrom Sephadex LH-20 column chromatography, while FIG. 3 shows thecorresponding chromatogram of Fraction 2 of the 60% methanol eluate.Both chromatograms were acquired using UV absorbance detection at 340nm. Fraction 1 exhibits well-resolved flavonoid peaks at R_(t) 29-48 minand includes some minor constituents of R_(t) 15-26 min. Fraction 2exhibits four peaks, two of which (R_(t) 39.4, 44.6) are predominant.

All peaks on the Fraction 1 chromatogram having retention time between29 and 48 min, peaks numbered from 1 to 22 as shown, display absorbanceprofiles corresponding to those of flavonols (Mabry et al. 1970; Machiexet al. 1990). Three peaks of R_(t) 34.3, 35.1 and 40.4 min were found tobe consistent with retention times and UV-visible spectra of standardsquercetin-3-galactoside (5), quercetin-3-glucoside (6) andquercetin-3-rhamnoside (11), respectively (FIG. 2, Table 1). The twomajor flavonoids that eluted in Fraction 2 (FIG. 3) were identified asmyricetin (& 39.8) and quercetin R_(t) 44.6).

Mass Spectrometry

APCI LC-MS analysis in the negative-ion mode was used to identify themolecular weights of constituents eluting in 60% methanol Fraction 1 andto ascertain whether they were sugar conjugates as evidenced by loss of162/132 mass units from the pseudomolecular ion. The results obtainedare summarized in Table 1 together with spectral characteristics,distribution of peaks by area percentages (at 340 nm) and retention timeunder chromatographic conditions employed. APCI LC-MS of phenolics givesintense deprotonated molecular ions [M−H]⁻ in the negative-ion mode.

Peak 1 exhibited an intense [M−H]⁻ ion peak at m/z 479 and a fragmention at m/z 317 [M-C₆H₁₁O₅]⁻ corresponding to a myricetin-hexoside. Thespectrum of the peaks 2 and 4 both gave [M−H]⁻ ion peaks at m/z 449 anda fragment ion at m/z 317 [M-C₅H₉O₄]⁻ consistent withmyricetin-pentoside conjugates. Peaks 5 and 6 exhibited in the APCI massspectra characteristic [M−H]⁻ ions at m/z 463 and fragment ions at m/z301 [M-C₆H₁₁O₅]⁻ corresponding to quercetin hexosides. These peaks, whencompared with chromatographic behavior and UV-visible spectra ofstandards, were identified as quercetin-3-β-galactoside andquercetin-3-β-glucoside, respectively.

Three peaks of R_(t) 36.3, 37.6, and 39.8 (8, 9, 10) showed intense[M−H]⁻ ion peaks at m/z 433 and fragment ions at m/z 301 [M-C₅H₉O₄]⁻consistent with quercetin-pentoside structures. The exact nature of thesugar moiety cannot be ascertained by LC-MS. The spectra released forpeak 11 exhibited a [M−H]⁻ ion at m/z 447 and a fragment ion at m/z 301[M-C₆H₁₁O₄]⁻ which corresponds to quercetin-3-α-rhamnoside as determinedby comparison with a standard.

Peak 12 gave a [M−H]⁻ ion peak at m/z 477 with a fragment ion at m/z 315[M-C₆H₁₁O₅]⁻ consistent with a possible structure containing amonomethoxyquercetin-hexoside. The position of the methyl substituentcould not be determined. Peaks 14 and 15 exhibited [M−H]⁻ ions at m/z447 and had fragment ions at m/z 315 [M-C₅H₉O₄]⁻ corresponding topossible methoxylated quercetin-pentosides. The MS spectra of peaks 13,16, 21 and 22 appear more complex and suggest the presence of a mixtureof components. Possible constituents of these peaks correspond tomonomethoxymyricetin-pentoside (m/z 463, 331) anddimethoxymyricetin-hexoside (m/z 507, 345) for peak 13 and toderivatives of methoxykaempferol (m/z 299) for peaks 21 and 22.Deprotonated molecular ions for peak 16 indicate the possible presenceof a monomethoxyquercetin-pentoside (m/z 447, 315) and an acylatedderivative of quercetin-hexoside (m/z 609, 301).

One of the latest eluting peaks at R_(t) 45.6, peak 19, gave [M−H]⁻ andfragment ion peaks at m/z 567 and 301 which we initially speculated tobe a quercetin-hexose ester with benzoic acid. Peak 19 was subsequentlyconfirmed to be quercetin-3-O-(6″-benzoyl)-β-galactoside by NMRspectroscopy.

NMR Spectroscopy

For structural determination of various flavonoid constituents by NMRspectroscopy, Fraction 1 of the 60% methanol eluate was furtherfractionated by preparative HPLC. The two step procedure applied for theelution of constituents with 60% methanol during Sephadex LH-20 columnchromatography was useful to prevent the coelution of the abundantsimple flavonols myricetin and quercetin with the peaks corresponding toflavonol conjugates (FIGS. 1 and 2). The NMR analysis of purified peakswas supported by ESI LC-MS data performed in the positive-ion mode(Table 1).

Sufficient amounts of eight pure components (2, 5, 6, 9, 10, 15, 16, 19)were obtained. These compounds were conclusively identified by NMR asmyricetin-3-β-xylopyranoside (2), quercetin-3-β-galactoside (5),quercetin-3-O-glucoside (6), quercetin-3-α-arabinopyranoside (9),quercetin-3-α-arabinofuranoside (10),3′-methoxyquercetin-3-α-xylopyranoside (15), andquercetin-3-O-(6″-benzoyl)-β-galactoside (19) (FIG. 4). Peak 16represented a mixture of coeluting compounds, and was subjected toadditional purification on a Sephadex LH-20 column with 60% methanol asthe eluting solvent. The compound with [M−H]⁻ ion peak at m/z 609 (16a)was used for structural determination by NMR spectroscopy, anddetermined to be quercetin-3-O-(6′-p-coumaroyl)-β-galactoside (FIG. 4).

The previous identification of peaks 5 and 6 asquercetin-3-β-galactoside and quercetin-3-β-glucoside, respectively,were confirmed by NMR data (Table 2). For peak 6, the anomeric protonappeared as a doublet (J=7.6 Hz) while the 2″, 3″, and 4″ protonresonances appeared as triplets (J=7.8-8.8 Hz), consistent with aglucose moiety. The flavonoid at R_(t) 39.8 (peak 10) was identified asquercetin-3-α-arabinofuranoside, which is consistent with previouslyreported data Puski et al. 1967; Yan et al. 2002).Quercetin-3-β-glucoside (6) and structures 2, 9, 15, 16a, and 19represent compounds not yet reported in cranberry or cranberry products.

Myricetin-3-β-xyloside (2) in the pyranose form was apparent due to the4″ carbon chemical shift and the strongly anisochronous 5″ methyleneresponses which are consistent with those of a pentose in the pyranoseform. The anomeric signal appeared as a J=7.43 Hz doublet, and takenalong with the downfield shift of the carbon resonance, indicates aβ-configuration. The 3″ proton resonance is an apparent triplet (J=8.66Hz), establishing a trans-diaxial relationship for the 2″-3″ and 3″-4″pairs.

The pyranose form of quercetin-3-α-arabinoside (9) was indicated by the4″ carbon chemical shift and the anisochronous 5″ methylene group. Theanomeric signal appeared as a J=5.2 Hz doublet at 5.22/102.3 ppm,implying the α-configuration. The 2″-3″ coupling constant of J=6.7 Hzindicated a trans-diaxial configuration, while 3″-4″ coupling constant(J=3.1 Hz) implies an axial-equatorial orientation, yielding theassignment of arabinopyranose. Thus, the relatively major flavonolquercetin-3-arabinoside exists in two sugar forms, as a pyranose (˜6.7%)and the previously reported (10) furanose form (˜9.7%) (Table 1).

Peak 15 presented NMR spectra indicative of the quercetin backbone and axylopyranose sugar moiety. An aromatic methoxy resonance was alsoobserved (3.71, 51.9 ppm) that yielded a 3-bond HMBC response to the 3′carbon resonance at ˜144 ppm, allowing assignment of the site of themethoxy substitution.

A quercetin backbone was observed in the isolate of peak 16a by NMRspectroscopy, and resonances consistent with the presence of agalactopyranose sugar moiety were observed. Two new AB spin systems wereobserved in the downfield region; one integrating for a total of twoprotons (7.58/145/5 and 6.29/114.6 ppm), the other integrating for fourprotons (7.46/130.9 and 6.99/116.7 ppm). The larger set of resonanceswas readily assigned as a 1,4-substituted aromatic system based on HMBCdata, while the smaller system was assigned as a trans-olefin due to its15.9 Hz coupling constant. An IMPRESS-HMBC data set was used toestablish an ester carbonyl linkage between the galactopyranose andolefinic moieties (Yang et al. 2003). These data, coupled with themolecular weight information, indicate that this isolate is the C-6″para-hydroxycinnamic acid ester of quercetin-3-β-galactopyranose (FIG.5).

Interpretation of the NMR spectra obtained on peak 19 (¹H reference,COSY, HSQC, HSQCTOCSY, and HMBC) revealed several structurallysignificant features. The resonances indicating a quercetin backbonewere readily observed and assigned by inspection. Resonances consistentwith a sugar moiety were also observed. The sugar resonances weresomewhat obscured by the residual water in the sample, but several keyassignments were possible. The anomeric methine doublet (J=7.6 Hz) wasobserved at 5.46/101.9 ppm, indicating an axial proton orientation, andtherefore a β-linkage to the quercetin backbone. COSY and HSQCTOCSYresponses allowed complete, sequential assignment of the sugar ring. Alarge coupling constant (J=8.3 Hz) was observed for the 2″-3″interaction, indicative of a trans-diaxial interaction, while the 4″proton resonance was observed as a broad singlet. This result requiresaxial-equatorial interactions for both 3″-4″ and 4″-5″ and yields theassignment of the sugar moiety as β-galactose.

Another spin system was identified in the aromatic region of thespectrum. This system consisted of an apparent doublet-triplet-tripletpattern in a 2:2:1 ratio and can be readily assigned as a phenyl group(ortho 7.621129.4; meta 7.26/129.3; para 7.49/133.9 ppm). Investigationof the HMBC data set revealed a 3-bond response between theortho-protons of this phenyl ring with a carbonyl resonance at 166.1ppm. A second 3-bond response to this same carbonyl carbon was observedfrom the 6″ methylene protons. Taken with the C-6″ chemical shift of64.9 ppm and the C-9″ chemical shift of 129.5 ppm, these data clearlyindicate the presence of a benzyl ester moiety at the 6″ position of thegalactose ring.

The cumulative data showed the presence of mostly glycosylated forms ofmyricetin and quercetin in Fraction 1 of the 60% methanol Sephadex LH-20eluate. Together with UV-spectra and peak area percentage these resultsconfirm the identity of peaks 4, 5, 10 and 11 asmyricetin-3-α-arabinofuranoside, quercetin-3-β-galactoside,quercetin-3-α-arabinofuranoside and quercetin-3-β-rhamnoside (Puski etal. 1967; Yan et al. 2002). The peaks labeled 1, 8 and 12 correspond tomyricetin-3-β-galactoside, quercetin-3-xyloside and3′-methoxyquercetin-3-β-galactoside (iso-rharmnetin-galactoside),respectively. These components were recently identified in cranberryextracts by Yan et al. (2002), however, the presence of other methylethers in cranberries has not been reported. We have fully characterizeda second methyl ether derivative in cranberries,3′-methoxyquercetin-3-α-xylopyranoside (15), and our LC-MS data suggestthe presence of a number of other methoxylated flavonols includingderivatives of quercetin, myricetin and kaempferol (Table 1).

EXAMPLE 2 Anti-Inflammatory Activity of Sephadex LH-20 Eluate Fractions

The anti-inflammatory activities of different classes of cranberryphenolic compounds separated by Sephadex LH-20 column chromatographywere tested in vivo with the 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced mouse ear edema assay described previously (Huang et al.2003). Each of the treatments was topically applied to both ears of fourfemale CD-1 mice. Treatments included application of either acetone orcurcumin controls or the test extract in 20 μl of acetone twenty minutesprior to application of either 5 mg of acetone alone or 1 nmol of TPA inacetone. At five hours post-treatment, mice were sacrificed and 6 mmdiameter ear punch biopsies were taken and weighed. Curcumin, awell-known anti-inflammatory agent, was used as a positive control (Chanet al. 1994; Chan 1995). Data were analyzed statistically using ANOVAand the Student-Neuman-Kuels (SNK) multiple means separation test(P≦0.05). The ratio of the difference in the weight of ear punchesbetween TPA treated groups receiving pretreatments of acetone and thetest extract, respectively, to the difference in the weight of earpunches between acetone pretreated groups subsequently treated with TPAand acetone, respectively, indicates the degree of anti-inflammatoryactivity of the test extract. Results are summarized in Table 3.

EXAMPLE 3 Anti-Inflammatory Activity ofQuercetin-3-O-(6″-benzoyl)-β-Galactoside

The anti-inflammatory activity ofquercetin-3-O-(6″-benzoyl)-β-galactoside (Peak 19) was tested in vivowith the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse earedema assay. Each of the treatments was topically applied to both earsof at least five CD-1 female mice. Treatments included application ofeither acetone or curcumin controls or the test extract in 20 μl ofacetone twenty minutes prior to application of 1.5 nmol of TPA inacetone. After 7 hours post-treatment, mice were sacrificed and 6 mmdiameter ear punch biopsies were taken and weighed. The ratio of thedifference in the weight of ear punches between TPA treated groupsreceiving pretreatments of acetone and the test extract, respectively,to the difference in the weight of ear punches between acetonepretreated groups subsequently treated with TPA and acetone,respectively, indicates the degree of anti-inflammatory activity of thetest extract. Results are summarized in Table 4.

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Zuo, Y., Wang, C., and Zhan J. Separation, characterization,quantitation of benzoic and phenolic antioxidants in American cranberryfruit by GC-MS. J. Agric. Food Chem. 2002, 50, 3789-3794. TABLE 1Retention Times, Relative Peak Areas, λ_(max), m/z values of [M + H]⁺,[M − H]⁻ and Fragment Ions of Flavonoid Constituents Resolved by HPLC of60% Methanol Fraction 1. Retention Relative time peak [M − H]⁻ andfragment ions [M + H]⁺ Peak (min) area, % λ_(max), nm in APCI MS in ESIMS Structure 1 29.0 8.3 261.5 356.5 479 (25%), 317 (100%) ndMyricetin-3-β-galactoside¹ 2 29.8 0.9 261.5 356.5 449 (100%), 317 (90%)451 Myricetin-3-α-xylopyranoside³ 3 32.0 2.2 233.2 266.2 nd nd nd 299.4356.5 4 32.5 3.6 261.5 356.5 449 (65%), 317 (100%) ndMyricetin-3-α-arabinofuranoside^(1,2) 5 34.3 21.5 256.8 356.5 463 (55%),301 (100%) 465 Quercetin-3-β-galactoside^(1,2) 6 35.1 0.9 256.8 356.5463 (60%), 301 (100%) 465 Quercetin-3-β-glucoside³ 7 35.8 1.3 256.8356.5 nd nd nd 8 36.3 8.2 256.8 356.5 433 (45%), 301 (100%) ndQuercetin-3-α-xylopyranoside¹ 9 37.6 6.7 256.8 356.5 433 (35%), 301(100%) 435 Quercetin-3-α-arabinopyranoside³ 10 39.8 9.7 256.8 356.5 433(35%), 301 (100%) 435 Quercetin-3-α-arabinofuranoside^(1,2) 11 40.4 14.3256.8 351.7 447 (55%), 301 (100%) nd Quercetin-3-rhamnopyranoside^(1,2)12 41.2 1.6 256.8 356.5 477 (100%), 315 (25%) nd3′-methoxyquercetin-3-β-galactoside¹ 13 41.7 3.9 256.8 356.5 507 (100%),463 (48%), nd Dimethoxymyricetin-hexoside 345 (48%), 331 (46%) ndMethoxymyricetin-pentoside 14 42.9 0.6 256.8 356.5 447 (100%), 315 (55%)nd Methoxyquercetin-pentoside 15 43.3 2.3 256.8 356.5 447 (100%), 315(32%) nd 3′-methoxyquercetin-3-α-xylopyranoside³ 16 44.0 4.6 256.8 351.7609 (35%), 447 (70%), 611⁴ Methoxyquercetin-pentoside;Quercetin-3-O-(6″-p- 256.8 318.4 315 (25%), 301 (100%)coumaroyl)-β-galactoside³ (Compound 16a) 351.7sh 17 44.5 0.8 256.8 356.5nd nd nd 18 44.8 1.5 256.8 313.6 nd nd nd 356.5 19 45.6 5.6 256.8 356.5567 (100%), 301 (52%) 569 Quercetin-3-O-(6″-benzoyl)-β-galactoside³ 2046.2 0.1 256.8 356.5 nd nd nd 21 46.8 0.3 256.8 356.5 581 (53%), 431(100%), nd Methoxykaempferol derivative 299 (28%) 22 47.4 1.1 256.8290sh 593 (50%), 431 (100%), nd Methoxykaempferol derivative 364.6 345(68%), 299 (75%)Note:nd—not determined;¹according to Yan et al. 2002 (10);²according to Puski et al. 1967 (9);³compounds not previously identified in cranberry;⁴[M + H]⁺ for peak 16a.

TABLE 2 ¹H and ¹³C NMR shifts of Cranberry Flavonol Glycosides (Partsper Million) 2 5 6 9 10 15 16a 19 Position ¹³C ¹H ¹³C ¹H ¹³C ¹H ¹³C ¹H¹³C ¹H ¹³C ¹H ¹³C ¹H ¹³C ¹H 6 99.5 6.15 99.4 6.04 100.4 6.06 99.6 6.1499.5 6.14 94.6 6.14 99.8 6.41 99.4 6.13 8 94.1 6.32 94.8 6.23 95.5 6.2794.5 6.35 94.3 6.35 89.7 6.39 94.6 6.54 94.0 6.29 2′ 109.1 7.12 117.17.59 116.8 7.50 116.8 7.47 116.1 7.43 108.6 7.71 117.0 7.84 116.5 7.455′ 116.1 6.79 116.1 6.81 116.1 6.80 116.2 6.79 111.0 6.82 116.4 7.05115.8 6.77 6′ 109.1 7.12 122.7 7.39 122.7 7.44 122.7 7.60 122.4 7.50118.2 7.46 122.7 7.85 122.8 7.58 1″ 102.5 5.31 103.1 4.97 102.2 5.06102.3 5.22 108.6 5.53 97.5 5.20 102.9 5.66 101.9 5.45 2″ 74.3 3.34 71.83.59 74.3 3.26 71.7 3.73 83.0 4.11 69.5 3.17 71.8 3.58 72.1 3.58 3″ 77.03.14 73.3 3.43 76.2 3.25 72.6 3.48 77.6 3.67 71.3 3.14 73.9 3.41 74.43.42 4″ 70.2 3.31 68.6 3.69 69.9 3.15 66.9 3.62 86.8 3.51 65.1 3.21 69.13.64 69.3 3.68 5″ 66.9 2.91/ 75.8 3.36 77.1 3.06 65.2 3.18/ 61.6 3.2561.7 3.54/ 73.8 3.66 74.2 3.75 3.60 3.57 2.89 6″ 60.9 3.34/ 61.1 3.34/64.3 4.26/ 64.9 4.23 3.44 3.47 4.35

TABLE 3 Inhibitory effect of cranberry fractions after Sephadex LH-20column chromatography on TPA-induced edema of mouse ears. Weight dataare expressed as the mean of 8 ear punches. MEAN WEIGHT SEPARATION OFEAR (SNK PUNCHES multiple means % TREATMENT (mg/Ear) test, P ≦ 0.05)INHIBITION Acetone + Acetone 7.4 e — Acetone + TPA 12.4 a — Cranberry,60% MeOH 10.7 b 34.3 (166 μg) + TPA Cranberry, 60% MeOH 8.5 c d e 78.6(500 μg) + TPA Cranberry, 100% MeOH 10.2 b c 45.3 (166 μg) + TPACranberry, 100% MeOH 9.8 b c d 53.3 (500 μg) + TPA Cranberry, 70%Acetone 8.6 c d e 77.0 (166 μg) + TPA Cranberry, 70% Acetone 8.1 d e85.5 (500 μg) + TPA Curcumin (95 μg) + TPA 8.5 c d e 79.0

TABLE 4 Inhibitory effect of quercetin-3-O-(6″-benzoyl)-β-galactoside(Cranberry peak 19) on TPA-induced edema of mouse ears. Weight data areexpressed as the mean of 10 ear punches (12 punches for Acetone + TPAtreatment.) ± the standard error. NUMBER WEIGHT OF OF MICE EAR PUNCHES %TREATMENT PER GROUP (mg/Ear) INHIBITION Acetone + Acetone 5  7.57 ± 0.13— Acetone + TPA 6 16.12 ± 0.64 — Cranberry Peak 19 5 13.21 ± 0.77 34.0(87.5 μg) + TPA Cranberry Peak 19 5 11.41 ± 0.54 55.1 (175 μg) + TPACurcumin 5 11.22 ± 0.77 57.3 (135 μg) + TPA

1. A flavonol composition comprising an extract of cranberry flavonols,the extract being substantially free of anthocyanins andproanthocyanidins, wherein each of the cranberry flavonols includespharmaceutically acceptable salts thereof.
 2. A method for theprevention and treatment of an inflammatory condition comprisingadministration of a therapeutically effective dose of the flavonolcomposition of claim
 1. 3. A pharmaceutical composition comprising apharmaceutically acceptable carrier in admixture with the flavonolcomposition of claim
 1. 4. A method for the prevention and treatment ofan inflammatory condition comprising administration of a therapeuticallyeffective dose of the pharmaceutical composition of claim
 3. 5. Adietary supplement composition comprising a consumable carrier inadmixture with the flavonol composition of claim
 1. 6. A method for theprevention and treatment of an inflammatory condition comprisingadministration of a therapeutically effective dose of the dietarysupplement composition of claim
 5. 7. A food composition comprising aconsumable carrier in admixture with the flavonol composition ofclaim
 1. 8. A method for the prevention and treatment of an inflammatorycondition comprising administration of a therapeutically effective doseof the food composition of claim
 7. 9. The food composition of claim 7wherein the consumable carrier is a cranberry-containing food product.10. The food composition of claim 9 wherein the cranberry-containingfood product is a dried cranberry, a sweetened and dried cranberry, apowdered cranberry, a flavored fruit piece, a sauce, a jelly, a relish,a juice, a wine or a cranberry juice-containing product.
 11. The foodcomposition of claim 7 wherein the consumable carrier is a beverage.12.-23. (canceled)
 24. A compound having the formula as shown, includingpharmaceutically acceptable salts thereof:


25. A method for the prevention and treatment of an inflammatorycondition comprising administration of a therapeutically effective doseof the compound of claim 24, including pharmaceutically acceptable saltsthereof.
 26. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier in admixture with the compound of claim
 24. 27. Amethod for the prevention and treatment of an inflammatory conditioncomprising administration of a therapeutically effective dose of thepharmaceutical composition of claim
 26. 28. A dietary supplementcomposition comprising a consumable carrier in admixture with thecompound of claim
 24. 29. A method for the prevention and treatment ofan inflammatory condition comprising administration of a therapeuticallyeffective dose of the dietary supplement composition of claim
 28. 30. Afood composition comprising a consumable carrier in admixture with thecompound of claim
 24. 31. A method for the prevention and treatment ofan inflammatory condition comprising administration of a therapeuticallyeffective dose of the food composition of claim
 30. 32. The foodcomposition of claim 30 wherein the consumable carrier is acranberry-containing food product.
 33. The food composition of claim 32,wherein the cranberry-containing food product is a dried cranberry, asweetened and dried cranberry, a powdered cranberry, a flavored fruitpiece, a sauce, a jelly, a relish, a wine or a cranberryjuice-containing product.
 34. The food composition of claim 30, whereinthe consumable carrier is a beverage.
 35. A cosmetic compositioncomprising a cosmetically acceptable carrier in admixture with theflavonol composition of claim
 1. 36. A method for the prevention andtreatment of an inflammatory condition comprising administration of atherapeutically effective dose of the cosmetic composition of claim 35.37.-38. (canceled)
 39. A cosmetic composition comprising a cosmeticallyacceptable carrier in admixture with the compound of claim
 24. 40. Amethod for the prevention and treatment of an inflammatory conditioncomprising administration of a therapeutically effective dose of thecosmetic composition of claim 39.